Exogenous artificial DNA forms chromatin structure with active transcription in yeast

Yeast artificial chromosomes (YACs) are important tools for sequencing, gene cloning, and transferring large quantities of genetic information. However, the structure and activity of YAC chromatin, as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events, have not been widely explored. Here, we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported data-carrying chromosome (dChr). In addition, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional, and proteomic characteristics of the exogenous dChr. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. The dChr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs. The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids. A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.

Multiscale mechanical models of DNA nanotubes: A theoretical basis for the design and tuning of DNA nanocarriers

DNA nanostructures are one of potential candidates for drug carriers due to their good biocompatibility and non-specificity in vivo. A reliable prediction about mechanical properties of artificial DNA structures is desirable to improve the efficiency of DNA drug carriers, however there is only a handful of information on mechanical functionalities of DNA nanotubes (DNTs). This paper focuses on quantifying the multiscale correlations among DNT deformation, packaging conditions and surrounding factors to tune mechanical properties of DNTs. By combining WLC statistical mechanics model, Parsegian's mesoscopic liquid crystal model and Euler's continuum beam theory, we developed a multiscale DNA-frame model; then theoretically characterize the initial packed states of DNTs for the first time, and reveal the diversity mechanism in mechanical properties of DNTs induced by interchain interactions and initial packed states. Moreover, the study of parameters, such as packaging conditions and environmental factors, provides a potential control strategy for tuning mechanical properties of DNTs. These conclusions provide a theoretical basis for accurately controlling the property and deformation of DNT in various DNT dynamic devices, such as DNA nanocarriers.

Polymer Chain Reaction (PCR): Principle and Applications

The new, standard molecular biologic system for duplicating DNA enzymatically devoid of employing a living organism, like E. coli or yeast, represents polymerases chain reaction (PCR). This technology allows an exponential intensification of a minor quantity of DNA molecule several times. Analysis can be straightforward with more DNA available. A thermal heat cycler performs a polymerization chain reaction that involves repeated cycles of heating and cooling the reactant tubes at the desired temperature for each reaction step. A heated deck is positioned on the upper reaction tube to avoid evaporating the reaction mixture (normally volumes range from 15 to 100 l per tube), or an oil layer can be placed on a reaction mixture surface. The amplified DNA fragment is determined based on selecting primers in addition to the starting and end of the DNA fragment. The primers stand for short, artificial DNA stripes, no higher than fifty (typically 18-25bp) nucleotides have been based on a starting and ending of DNA fragment to be amplified. DNA-polymerase connects and starts a new DNA strand synthesis The PCR products can be visualized by dual foremost methods: (1) staining of the product of DNA amplified by a chemical dye like bromide ethidium, or (2) marking of fluorescent dyes (fluorophores) PCR primers or nucleotides before amplification of PCRs. PCR offers some benefits. First, it is a simple method of understanding and using and quick results. It has an extremely sensitive technology with the potential for sequencing, cloning, and analyzing millions or milliards of copies of a particular product.

Plasmid hypermutation using a targeted artificial DNA replisome

Extensive exploration of a protein’s sequence space for improved or new molecular functions requires in vivo evolution with large populations. But disentangling the evolution of a target protein from the rest of the proteome is challenging. Here, we designed a protein complex of a targeted artificial DNA replisome (TADR) that operates in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of the target plasmid up to 2.3 × 105–fold with only a 78-fold increase in off-target mutagenesis. It was used to evolve itself to increase error rate and increase the efficiency of an efflux pump while simultaneously expanding the substrate repertoire. TADR enables multiple simultaneous substitutions to discover functions inaccessible by accumulating single substitutions, affording potential for solving hard problems in molecular evolution and developing biologic drugs and industrial catalysts.

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers

1. The study of environmental DNA released by aquatic organisms in their habitat offers a fast, non-invasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as filtration and precipitation are time consuming, require human intervention and are not applicable to a wide range of habitats such as turbid waters and poorly-accessible environments. To circumvent these limitations, we propose to use the binding properties of minerals to create a passive eDNA sampler. 2. We have designed 3D-printed samplers made of hydroxyapatite (HAp samplers), a mineral known for its high binding affinity with DNA. The shape and the geometry of the samplers have been designed to facilitate their handling in laboratory and field. Here we describe and test the ability of HAp samplers to recover artificial DNA and eDNA. 3. We show that HAp samplers efficiently recover DNA and are effective even on small amounts of eDNA (<1 ng). However, we also observed large variations in the amount of DNA recovered even under controlled conditions. 4. By better understanding the physico-chemical interactions between DNA and the HAp sampler surface, one could improve the repeatability of the sampling process and provide an easy-to-use eDNA sampling tool for aquatic environments.

Development of new pathogen surrogates and synthetic DNA tracers for water applications

&lt;p&gt;In recent years, we have conducted research into developing new pathogen surrogates and synthetic DNA tracers for water applications. Biomolecule-modified particles have been used to mimic &lt;em&gt;Cryptosporidium&lt;/em&gt;, rotavirus and adenovirus with respect to their filtration removal and transport in porous media. Additionally, we have developed new DNA tracers as free DNA molecules or DNA-encapsulated biopolymer microparticles to track water contamination. DNA markers are also used to label some surrogates to facilitate their sensitive detection by using qPCR.&lt;/p&gt;&lt;p&gt;The surrogates have been validated in laboratory conditions alongside the actual pathogens. The &lt;em&gt;Cryptosporidium &lt;/em&gt;surrogates have been satisfactorily validated in alluvial sand, in limestone sand, in coagulation and rapid sand filtration studies. The rotavirus surrogates have been successfully validated in coastal sand aquifer media, in unmodified and hydrophobically modified quartz sand, and in stony alluvial soils under on-site wastewater applications. The research findings have demonstrated that these new surrogates significantly outperform the most commonly used existing surrogates, namely, unmodified microspheres for &lt;em&gt;Cryptosporidium &lt;/em&gt;oocysts and MS2 phage for viruses. Working with the water industry, we have applied the &lt;em&gt;Cryptosporidium &lt;/em&gt;surrogate to pilot-scale rapid sand filters and point-of-use domestic filters and determined its removal efficiencies in water filtration systems commonly used in New Zealand. The artificial DNA tracers have been validated in surface water, groundwater and soils, and they were readily trackable in a surface stream for up to 1 km.&lt;/p&gt;&lt;p&gt;Our proof-of-concept studies suggest that the new pathogen surrogates and synthetic DNA tracers we have developed show great promise as new tools for water applications. The &amp;#8216;micro mimics&amp;#8217; approach has opened up a new avenue for assessing pathogen removal and transport in water systems without the risk and expense that accompany work with actual pathogens. With further validation, the new surrogates could be used to study pathogen removal and transport in subsurface media after the disposal of effluent and biosolids to land, and to assess the performance of filtration processes in water and wastewater treatment. With future up-scaling validation of the new synthetic DNA tracers, these tracers could be useful for concurrently tracking multiple pollution sources and pathways in freshwater environments.&lt;/p&gt;

Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase–hexahistidine–2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.

Multiple Alignment of Promoter Sequences from the Arabidopsis thaliana L. Genome

In this study, we developed a new mathematical method for performing multiple alignment of highly divergent sequences (MAHDS), i.e., sequences that have on average more than 2.5 substitutions per position (x). We generated sets of artificial DNA sequences with x ranging from 0 to 4.4 and applied MAHDS as well as currently used multiple sequence alignment algorithms, including ClustalW, MAFFT, T-Coffee, Kalign, and Muscle to these sets. The results indicated that most of the existing methods could produce statistically significant alignments only for the sets with x < 2.5, whereas MAHDS could operate on sequences with x = 4.4. We also used MAHDS to analyze a set of promoter sequences from the Arabidopsis thaliana genome and discovered many conserved regions upstream of the transcription initiation site (from −499 to +1 bp); a part of the downstream region (from +1 to +70 bp) also significantly contributed to the obtained alignments. The possibilities of applying the newly developed method for the identification of promoter sequences in any genome are discussed. A server for multiple alignment of nucleotide sequences has been created.